LYSINE-BASED STRUCTURE IN THE PROREGION OF PROCATHEPSIN-L IS THE RECOGNITION SITE FOR MANNOSE PHOSPHORYLATION

The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes, It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved, Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation, The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation, Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L, By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction, On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site, In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.